Hepatocyte growth factor activator (HGFA) is a plasma protein secreted mainly by the liver that regulates the mitogenic, motogenic, and morphogenic activities of hepatocyte growth factor (HGF, also known as scatter factor (SF)). (Shimomura et al., Cytotech., 8:219-229 (1992)). HGF is implicated in embryonic development, tissue regeneration and invasive tumor growth. This activity requires proteolytic processing of HGF into a two-chain, disulfide-linked α,β-heterodimeric form. HGFA is among the most potent activators of HGF identified so far. (Shimomura et al., Eur. J. Biochem. 229 (1995)). HGFA expression has been reported in normal gastrointestinal renal tissues, and in the central nervous system, as well as in pancreatic, hepatocellular, colorectal, prostatic, and lung cancer cells. (Itoh et al., Biochim. Biophys. Acta, 1491:295-302 (2000); van Adelsberg et al., J. Biol. Chem., 276:15099-15106 (2001); Hayashi et al., Brain Res., 799:311-316 (1998); Moriyama et al., FEBS Lett., 372:78-82 (1995); Parr et al., Int. J. Oncol., 19:857-863 (2001); Kataoka et al., Cancer Res., 60:6148-6159 (2000); Nagata et al., Biochem. Biophys. Res. Comm., 289:205-211 (2001)). Recently, HGFA secretion from multiple myeloma cells has been linked to the potent para- and/or autocrine effect of HGF. (Tj in et al., Blood, 104:2172-2175 (2004)).
HGFA is secreted as a 96 kDa zymogen (proHGFA) with a domain structure like that of coagulation factor XII (FXIIa), comprising 6 domains. Those domains include an N-terminal fibronectin type II domain, an epidermal growth factor (EGF)-like domain, a fibronectin type 1 domain, another EGF-like domain, a kringle domain, and a C-terminal trypsin homology serine protease domain. (Miyazawa, et al., J. Biol. Chem., 268:10024-10028 (1993)). Cleavage at a kallikrein-sensitive site between residues Arg372 and Val373 can produce a short 34 kDa form that lacks the first 5 domains. Both the 96 kDa and 34 kDa forms of proHGFA can be cleaved between residues Arg407 and Ile408 into active HGFA by thrombin. (Shimomura et al., J. Biol. Chem., 268, 22927-22932 (1993)). Thrombin is the ultimate effector of pro-coagulant stimuli and generation of active HGFA would be consistent with the activity of HGF in wound repair. (Bussolino et al., J. Cell Biol., 119:625-641 (1992)).
Among factors influencing HGF/Met signaling are the activation of proHGFA and subsequent inhibition of HGFA. The identified physiological inhibitors of HGFA are the splice variants HAI-1 and HAI-1B (hepatocyte growth factor activator inhibitor-1), and HAI-2 (also known as placental bikunin). (Shimomura et al., J. Biol. Chem., 272:6370-6376 (1997); Kawaguchi et al., J. Biol. Chem., 272:27558-27564 (1997); Kirchhofer et al., J. Biol. Chem. 278:36341-36349 (2003)). HAI-1 and HAI-1B (collectively referred to as HAI-1/B) are expressed in tissues and in cells at the same levels and are identical, except that HAI-1B has an additional 16 amino acids between the first and second Kunitz domains. (Kirchhofer et al., cited supra). HAI-1B and HAI-2 are membrane-anchored proteins and, despite significant differences in size and domain organization, each have two Kunitz domains (KD). In each protein, the first KD (KD 1) has been shown to be responsible for inhibition of human HGFA. (Kirchhofer et al., cited supra; Denda et al., J. Biol. Chem., 277:14053-14059 (2002); Qin, et al., FEBS Lett. 436:111-114 (1998)). Membrane sequestration of HAI-1B and HAI-2 is consistent with the observed activity of HGFA in serum. However, membrane shedding of the HAI-1 extracellular domain has been reported to produce soluble long (˜58 KDa) and short (˜40 KDa) forms from cleavage at two distinct sites, with the long form exhibiting low affinity for HGFA. (Shimomura et al., J. Biochem., 126, 821-828 (1999)). This finding suggests an additional mechanism regulating HGF/Met signaling. (Kataoka et al., Cancer and Metastasis Rev., 22:223-236 (2003)).
One hundred-eighteen human serine protease genes with trypsin homology have been identified in the human genome, having known hydrolytic functions in systems as diverse as food digestion and blood coagulation. (Lander et al., Nature, 409:860-921 (2001)). Structural biology in the trypsin/chymotrypsin system dates to the era before facile production of proteins using recombinant DNA. (Huber et al., Acc. Chem. Res., 11: 114-122 (1978)). Some aspects of the active site conformations of serine proteases have been identified including the arrangement of the catalytic triad (His, Asp and Ser), the presence of an oxyanion hole that stabilizes the transition state, and provision for substrate binding in a cleft that provides both general and specific interactions. However, some enzyme active sites have proven conformationally labile under the influence of specific binding partners. (Schmidt et al., J. Thromb. Haemost., 1, abstract OC448 (2003)). For example, a small molecule inhibitor of coagulation factor VIIa, G17905, induces an unconventional arrangement of the oxyanion hole. (Olivero, J. Biol. Chem., submitted (2004)).
On the other hand, the conformational state of trypsin-like active sites without substrate or with a substrate-like inhibitor is much less well known. (Cavarelli et al., Structure, 5:813-824 (1997); Vath et al., Biochemistry 36, 1559-1566 (1997); Vath et al., Biochemistry, 38:10239-10246 (1999); Papageorgiou et al., Prot. Sci., 9:610-618 (2000)). The structures of exfoliative toxin A (ETA) and exfoliative toxin B (ETB) structures suggest a low energy barrier exists between the conventional active site and one with the inverted oxyanion hole like that seen for G17905/FVIIa. Also, FVIIa with and without the small molecule inhibitor, benzamidine, differ in a small rotation of the Ser214-Trp215 peptide bond and increased thermal factors. (Sichler et al., J. Mol. Biol., 322:591-603 (2002)). A distinct category of unoccupied active site conformations is presented by α1-tryptase. (Marquart et al., J. Mol. Biol., 321:491-502 (2002)). Unlike their close homologues the β-tryptases, α-tryptases are essentially not active for the hydrolysis of tested substrates. It is thought that the low activity is due to a Asp substitution of Gly216 in the substrate binding cleft, and the recent X-ray structure has revealed a kink in the important 214-220 segment. (Marquart et al., cited supra). There are also reports of altered active site conformations arising in true enzymes without a substrate-like inhibitor when there is an additional influence from the absence of a cofactor, for instance thrombin without Na+, or from mutations. (Pineda et al., J. Biol. Chem., 277:40177-40180 (2002); Pineda et al., J. Biol. Chem., in press (2004); Szabo et al., Eur. J. Biochem., 263:20-26 (1999)). Additional conformational variations of the protease active site regions come from non-enzymatic homologues. Some proteins with easily identifiable trypsin homology are, in fact, not hydrolases. For instance, the recent X-ray structure of the HGF protease-like domain revealed a pseudo-active site in which normal substrate binding is not possible. (Kirchhofer et al., J. Biol. Chem., 279:39915-39924 (2004)).
There remains a need to develop new therapeutics useful to treat cancer and other diseases associated with HGF/Met signaling. Control over HGF signaling may provide a valuable therapeutic benefit in cancer or other diseases associated with HGF/Met signaling. HGFA is an activator of HGF activity and thus, modulation of HGFA activity can affect HGF signaling.